Lecture 17 Analysis of Gene Expression - Rutgers...
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Lecture 17 Analysis of Gene Expression 1) Reporter gene assay 2) DNase footprinting assay 3) RNase protection assay 4) Gel-retardation assay 5) Filter paper binding assay 6) Nuclear run-on and run-off assay
Transcript of Lecture 17 Analysis of Gene Expression - Rutgers...
Lecture 17Analysis of Gene Expression
1) Reporter gene assay
2) DNase footprinting assay
3) RNase protection assay
4) Gel-retardation assay
5) Filter paper binding assay
6) Nuclear run-on and run-off assay
Reporter Gene Assay
1) Join the regulatory sequence of interest to a reporter geneor some marker carried in an expression vector.
2) The recombinant construct is introduced into the cell andthe activity of the regulatory sequence (i.e. promoteractivity) can be determined.
3) Reporter genes or markers provide a convenient means toidentify and analyze the regulatory elements of genes.
4) Reporter system measures the transcriptional activity (theinteraction of the cis-elements on the promoter with thetrans-acting factors).
Chloramphenicol acetyl-transferase (CAT) assay
CAT assay: promoter activity is expressed by thedegree of acetylation of chloramphenicol
Gorman et al., 1982 MCB 2, 1044-1051For more detail: www.promega.com
Luciferase as the reporter gene
Luciferase as the reporter gene
Luciferase as the reporter gene
Firefly beetle ( Photinus pyralis) luciferase is one of the mostpopular reporter molecules used in molecular biology andbiochemistry (Gould & Subramani, 1988; Vieites et al., 1994;Gailey et al., 1997).
Luciferase can be used to monitor promoter response activityin bacteria, cultured cells, and transgenic plants or animals.
By providing faster results, lower costs, and over a 1,000-foldincrease in sensitivity, the luciferase assay has largelyreplaced the standard 14C chloramphenicol acetyl-transferase(CAT) assay.
Luciferase assay
Stratagene Pathway Profiling System
pSEAP2-Basic allows for expression of secreted alkaline phosphatase (SEAP).
SEAP reporter gene
SEAP reporter gene
CSPD: [3-(4-methoxyspiro[1,2-dioxetane-3,2’(5’-chloro)-tricyclo(3.3.1.13,7) decane]-4-yl)phenyl phosphate]
SEAP reporter gene
SEAP reporter gene
AmershamPharmaciaBiotech(800) 526-3593www.apbiotech.com
AppliedBiosystems(800) 345-5224www.appliedbiosystems.com
BD Biosciences-CLONTECH(800) 662-2566www.clontech.com
BD Biosciences-Pharmingen(877) 232-8995www.bdbiosciences.com
Bio Rad(800) 4-BIORADwww.biorad.com
Bio Vision Inc.(800) 891-9699www.biovisionlabs.com
Gene TherapySystems Inc.(888) 428-0558www.genetherapysystems.com
ICN(800) 854-0530www.icnbiomed.com
Intergen(800) 431-4505www.intergenco.com
Invitrogen(800) 955-6288www.invitrogen.com
InvivoGen(888) 457-5873www.invivogen.com
Marker GeneTech. Inc.(541) 342-3760www.markergene.com
MolecularDevices(800) 635-5577www.moleculardevices.com
MolecularProbes(541) 465-8300www.probes.com
Novagen(800) 526-7319www.novagen.com
Packard BioScience Co.(800) 323-1891www.packardbioscience.com
PanVera(800) 791-1400www.panvera.com
PerkinElmer Life Sciences(800) 551-2121lifesciences.perkinelmer.com
Pierce Chemical Co.(800) 874-3723www.piercenet.com
Promega(800) 356-9526www.promega.com
Roche MolecularBiochemicals(800) 262-1640biochem.roche.com
Stratagene(800) 894-1304www.stratagene.com
ThermoLabsystems(508) 520-0009www.labsystems.fi
Suppliers of Reporter Assay Systems
Footpriting assay on DNA-protein interaction
DMS footprinting assay
1) Isolation of RNA sample(s) to be examined for target expression2) Synthesis of a labeled antisense RNA probe complementary to a several-hundred-base region of the target mRNA,3) Hybridization of the labeled probe to a total RNA sample,4) Treatment of the sample with single-strand-specific RNase to degrade unhybridized probe and target5) Separation of the remaining protected probe::target hybrids on a denaturing polyacrylamide gel6) Detection/quantitation of the RNase-resistant "protected" probe
RNase Protection Assay
Multi-probe RPA systems : a series of apoptosis-related gene templates, each of distinct length and eachrepresenting a sequence in a distinct mRNA species.T7 polymerase-directed synthesis of a high-specific-activity, [32P]-labeled antisense RNA probe set.The probe set is hybridized in excess to target RNA in solution after which free probe and other single-stranded RNA are digested with RNases.The remaining "RNase-protected" probes are purified, resolved on denaturing polyacrylamide gel, andquantified by autoradiography or phosphorimaging.The quantity of each mRNA species in the original RNA sample can then be determined based on theintensity of the appropriately-sized, protected probe fragment.
Two distinct advantages of the multi-probe RPA approach are its sensitivityand its capacity to simultaneously quantify several mRNA species, in a singlesample of total RNA.
This allows comparative analysis of different mRNA species within samplesand, by incorporating probes for housekeeping gene transcripts, the levels ofindividual mRNA species can be compared between samples.
Moreover,the assay is highly specific and quantitative due to the RNasesensitivity of mismatched base pairs and the use of solution-phasehybridization driven toward completion by excess probe.
Lastly, the multi-probe RPA can be preformed on total RNA preparationsderived by standard methods from either frozen tissues or cultured cells,without further purification of poly-A+ RNA.
RNase Protection Assay
bclx
p53GADD45c-fosp21bax
bcl-2
mcl1
L32
GAPDH
0 4 16 24 4 16 24Time (hr)
Probe
0 4 16 24 4 16 24 - + - +R-3
WI38 WI38VA
RNase Protection Assay
Simultaneous Quantitation of Multiple mRNAs
Gel mobility shift assay (GMSA)
Detect interaction between a protein and DNA by the retardation of theelectrophoretic mobility of DNA that occurs upon binding to the protein
Gel mobility shift assay
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
BSA 18klys 18Khyp BSA 18klys 18Khyp
Random Pool RNA Post-SELEX RNA
Systematic Evolution of Ligands by EXponential enrichment
11 clones post-SELEX RNA, all bind to eIF5A in hypusine-dependent manner
RNA
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Poly(A)
6xHis-eIF5A0 6xHis-eIF5Adh HeLa eIF5A
6xHi
s-eI
F5A
hyp
2.5 5 7.50.6 1.3 2.5 5
0 0.6 1.3 2.5 5 7.5
2.2 µg
RNA binding assayRNA
EMSA with staining
Filter binding assayNitrocellulose membrane filters bind ssDNA, protein, but not dsDNA.However, if dsDNA binds to a protein, the complex will bind to the filter.This forms the basic principle of using nitrocellulose filter to determinethe binding constant and binding activity
Nuclear run-on transcription assayA way of ascertaining which genes are active in a given cell by allowing transcription ofthese genes to continue in the isolated nuclei. Specific transcripts can be identified bytheir hybridization to known DNAs on dot blots.
Nuclear run-off transcription assay
It is a means ofchecking the efficiencyand accuracy of in vitrotranscription.
DNA fragment containing a gene is truncated inthe middle. Transcription is performed in vitro.The length of the transcript allows us to locatethe transcription start site, and the amount of thetranscript reflects the efficiency of transcription.
